48 February / March 2020
Figure 1.
Comparison of a full mass spectra over time between a plasma sample prepared using protein precipitation (A) and that prepared using solid phase extraction (B). Areas of detected ions are circled.
Figure 1 demonstrates the effect that different sample preparation techniques can have. This figure looks at the full scan spectra of a blank matrix extracted either using protein precipitation or by using solid phase extraction as a function of time with the intensity of a particular mass being highlighted by the intensity of the colour. The protein precipitation is performed using 3:1 acetonitrile to blank rat plasma (100 µL), whereas the solid phase extraction utilises a polymeric stationary phase, and washing with 30% methanol in water and eluting with 100% methanol. For the SPE method the 100 µL blank rat plasma was added to 900 µL of water prior to addition to the 1 mL 30 mg cartridge.
The chromatography was obtained on a C18 column. The mobile phases were 0.1% formic acid in water [A] and 0.1% formic acid in methanol [B]. A gradient program was used in the elution of the analytes from the column; 95% [A] and 5% [B] for 0.5 min, linear change to 5% [A] and 95% [B] over 3 min and hold for 1 min, then revert back to 95% [A] and 5% [B] and hold for 0.5 min. The flow rate was 0.6 mL/min, with the injection volume of 10 µL.
It can be seen that the protein precipitation results in a higher background level of ions, which would not be observed with many traditional assays that focus on a single parent daughter transition and do not look at a full scan spectra. In particular, there is a higher intensity of ions at longer elution times and also at the beginning of the chromatogram. Figure 2 highlights another issue with the protein precipitation approach in that it takes several aqueous blank injections before the matrix is removed. An interesting observation is that the mass spectra obtained with the first aqueous blank has higher molecular masses eluting when compared to the plasma extracted sample [11].
In the previous scenario the use of SPE would be beneficial to improving the robustness of the assay; however the use SPE does require a degree of dexterity to ensure that optimal performance is maintained. Figure 3 highlights one of the issues associated with SPE and one that can be quite common when dealing with multiple samples being processed simultaneously, either on a SPE manifold or using a 96 deep well (DW96) plate format. In both of these scenarios it is not uncommon to have different flow rates in different tubes/wells. There are a variety of reasons why this might exist; from poor manufacture of the SPE frits (pore structure variability etc.), to variations in the samples that are being tested, resulting in very different inter tube/well flow rates being experienced during the sample preparation step which can affect the recovery. Figure 3 shows an elution profile obtained from two
Figure 2
Comparison of full mass spectra over time for 1st, 5th and 9th aqueous injec- tions subsequent to a protein precipitated sample.
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